Epicatechin modulates stress-resistance in C. elegans via insulin/IGF-1 signaling pathway

Abstract

The nematode Caenorhabditis elegans has been used to examine the influence of epicatechin (EC), an abundant flavonoid in the human diet, in some stress biomarkers (ROS production, lipid peroxidation and protein carbonylation). Furthermore, the ability of EC to modulate the expression of some key genes in the insulin/IGF-1 signaling pathway (IIS), involved in longevity and oxidative or heat shock stress response, has also been explored. The final aim was to contribute to the elucidation of the mechanisms involved in the biological effects of flavonoids. The results showed that EC-treated wild-type C. elegans exhibited increased survival and reduced oxidative damage of biomolecules when submitted to thermal stress. EC treatment led to a moderate elevation in ROS levels, which might activate endogenous mechanisms of defense protecting against oxidative insult. The enhanced stress resistance induced by EC was found to be mediated through the IIS pathway, since assays in daf-2, age-1, akt-1, akt-2, sgk-1, daf-16, skn-1 and hsf-1 loss of function mutant strains failed to show any heat-resistant phenotype against thermal stress when treated with EC. Consistently, EC treatment upregulated the expression of some stress resistance associated genes, such as gst-4, hsp-16.2 and hsp-70, which are downstream regulated by the IIS pathway.

Fig 1. Scheme of C. elegans IIS pathway.

Fig 2

Percentages of survival following thermal stress (35°C, 8h) applied at days 10^th (A) and 17^th of adulthood (B) in N2 wild type C. elegans strain not treated (controls) and treated with EC (200 μM in the culture media). Three independent experiments were performed. The results are presented as the mean values ± SD. Statistical significance was calculated using the Chi Square Test. The differences were considered significant at *(p<0.05).

Fig 3

Levels of intracellular ROS in C. elegans subjected (B) or not (A) to thermal stress (35°C, 2h) grown in the absence (controls) or presence of EC (200 μM in the culture media). ROS levels were evaluated at different ages throughout the entire life of the worm. Five independent experiments were performed. The results are presented as the mean values ± SEM. Statistical significance was calculated using one-way analysis of variance ANOVA. The differences were considered significant at *(p<0.05).

Fig 4

Levels of (A) carbonylated proteins and (B) lipid peroxidation products after cultivation of C. elegans in the absence (controls) and presence of EC (200 μM) and subjected to thermal stress. The results were obtained at days 10^th (A.1 and B.1) and 17^th (A.2 and B.2) of worm adulthood. Three independent experiments were performed. The results are presented as the mean values ± SEM. Statistical significance was calculated using one-way analysis of variance (ANOVA). The differences were considered significant at *(p<0.05).

Fig 5. Percentages of survival following thermal stress applied at days 2^nd and 9^th of adulthood in different long-lived C. elegans mutants from the IIS pathway cultivated in the absence (controls) and presence of EC (200 μM) in the culture media.

Three independent experiments were performed. The results are presented as the mean values±SD. Statistical significance was calculated using the Chi Square Test. The differences were considered significant at *(p<0.05).

Fig 6

Percentage of survival following thermal stress applied at days 2nd (A, C and D) and 9th (B, D and F) of adulthood in daf-16(mu86), hsf-1(sy441) and skn-1(zu67) mutants cultivated in the absence (controls) and presence of EC (200 μM) in the culture media. Three independent experiments were performed. The results are presented as the mean values±SD. Statistical significance was calculated using the Chi Square Test. D The differences were considered significant at *(p<0.05).

Fig 7

Effect of EC on the expression of daf-16, hsf-1 and skn-1 genes in wild-type C. elegans cultivated in the absence (controls) and presence of EC (200 μM) in the culture media grown under non-stressed conditions (A) or after subjecting them to thermal stress (B). The expression level was determined by RT-qPCR; act-1 was used as an internal control. Nine independent experiments were performed. The results are presented as the mean values ± SEM. Statistical significance was calculated using by one-way analysis of variance ANOVA The differences were considered significant at (*p<0.05).

Fig 8. Effect of EC on the expression of gst-4, sod-3, hsp-16.2 and hsp-70 in C.elegans.

Age-syncronized L1 transgenic worms of Pgst-4::gfp, Psod-3::gfp, Phsp-16.2::gfp and Phsp-70::gfp reporter strains were cultivated in the absence (controls) and presence of EC (200 μM) in the culture media. A) Representative fluorescence images of control and EC-treated worm strains stress response. B) Relative fluorescence intensities of transgenic worms. Total GFP fluorescence of each whole worm was quantified using Image J sofware. Three independent experiments were performed. The results are presented as the mean values ± SEM. Approximately 35 ramdomly selected worms from each set of experiments were examined. Differences compared with the control (0 μM, 0.1% DMSO) were considered statistically significant at p<0.05 (*) and p<0.01 (**) and p<0.001 (***) by one-way ANOVA.

Fig 9. Effect of EC on DAF-16::GFP nuclear localization.

Transgenic worms expressing the fusion protein DAF-16::GFP were cultivated in the absence (controls) and presence of EC (200 μM) and evaluated at 2^nd day of adulthood. DAF-16::GFP subcellular distribution was classified as cytosolic, intermediate and nuclear.

Fig 10. Effect of EC on the expression of gst-7 and ctl-1 genes in wild-type C. elegans cultivated in the absence (controls) and presence of EC (200 μM).

The expression level was determined by RT-qPCR; act-1 was used as an internal control. Nine independent experiments were performed. The results are presented as the mean values ± SEM. Statistical significance was calculated using by one-way analysis of variance ANOVA. The differences were considered significant at (*p<0.05).